We have shown that the ts lesion in the mouse mammary tumor cell line FT210 is located in CDC2 gene, the mammalian homologue of the S. pombe cdc2 gene known to be a key regulatory gene in the yeast cell division cycle. Two point mutations have been identified in the CDC2 gene resulting in amino acid replacements in conserved regions of the p34 kinase. One of these replacements is in the rigidly conserved PSTAIR region. Other laboratories have shown that injection of just the PSTAIR containing peptide induces major changes in cell cycle events. We have rescued FT2 10 cells with the human CDC2 gene and isolated 6 stably transformed FT2Hs cells lines. Four H1 kinase activities have been isolated, three of which contain p34 and are thermolabile. We propose to study the role of the conserved PSTAIR peptide in cell cycle progression, particularly through G1/S and G2/M. Unlike all other cdc2 mutations studies in S. pombe, the mutation in the CDC2Mm gene affects progression through only G2/M. The cdc2 S. pombe gene will be modified to reflect the same amino acid substitution found in the FT210 CDC2Mmts gene and, by direct replacement of the endogenous yeast gene, the mutation in the PSTAIR region will be tested for its effect on cellular progression through the yeast cell cycle. The four Hl kinase activities will bc fully characterized by the identification of their molecular components and the sites of phosphorylation of their H1 substrates. Affinity purification techniques, particularly those applicable to p34 kinases, will be employed to obtain highly purified H1 kinase fractions. The cell cycle behavior of the H1 kinases as related to G1, S, and G2/M phosphorylation of the H1 histones will be studies in synchronized FT210 cells. Although G2/M hyperphosphorylation of Hl has been correlated with the process of chromosome condensation, cause and effect have yet to be demonstrated. DNA supercoiling assays have now been established and we propose to determine the effects of HI hyperphosphorylation on DNA topology. The HI kinase activities will be tested for their ability to induce compaction of chromatin in the homogenous and highly defined supercoiling assay, in in vitro assays using native chromatin, and in situ with nuclei isolated from temperature arrested G2-phase FT210 cells. The effects of H1 phosphorylation on chromatin structure will be assessed by monitoring alterations in the solubility of the chromatin, electron microscopy, low-angle x-ray and neutron scatter of chromatin, and by DNA staining of nuclei substrates.